The HNK-1 carbohydrate epitope in the eye: basic science and functional implications.

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules. It has been implicated in cell to cell and cell to extracellular matrix adhesion, and antibodies to the HNK-1 epitope are emerging as a versatile tool in eye research. They have been used to identify a novel cell type in the human eye, the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in its antigenic profile from fibroblasts of other tissues. These cells are associated with the elastic fiber system of the ICTL. Other structures in the human eye that harbor the HNK-1 epitope in a nonrandom pattern are the ciliary and iris epithelia, the zonular lamella, the lens capsule, the retina, glial cells of the optic and ciliary nerves, and scleral fibroblasts. The HNK-1 epitope in the eye appears early during embryonic development and is phylogenetically conserved, but many interspecies differences exist in its distribution. The role of the HNK-1 epitope may be to structurally stabilize the ciliary body and the retina, and to participate in zonular attachments. The HNK-1 epitope has been linked with many common eye diseases. The subepithelial matrix cells seem to be susceptible to undergo irreversible damage as a result of glaucoma, thermal injury, and tissue compression. This epitope has proved to be useful in identifying intraocular deposits of exfoliation syndrome. It can explain the adhesiveness of exfoliation material. Intraocular exfoliation material differs in HNK-1 immunoreactivity from the extraocular fibrillopathy of exfoliation syndrome and its presence in fellow eyes also argues against the concept of unilateral exfoliation syndrome. The HNK-1 epitope is found in the extracellular matrix of secondary cataract and anterior subcapsular cataract, and it may contribute to their pathogenesis. Finally, the HNK-1 epitope can be used to trace neuroepithelial derivatives of the optic vesicle in developmental anomalies and in tumors of the eye. Eventual identification of molecules that bear the HNK-1 epitope in the eye will likely shed light on many aspects of ocular physiology and pathobiology

Structure, development and function of cytoskeletal elements in non-neuronal cells of the human eye.

The cytoskeleton, of which the main components in the human eye are actin microfilaments, intermediate filaments and microtubules with their associated proteins, is essential for the normal growth, maturation, differentiation, integrity and function of its cells. These components interact with intra- and extracellular environment and each other, and their profile frequently changes during development, according to physiologic demands, and in various diseases. The ocular cytoskeleton is unique in many ways. A special pair of cytokeratins, CK 3 and 12, has apparently evolved only for the purposes of the corneal epithelium. However, other cytokeratins such as CK 4, 5, 14, and 19 are also important for the normal ocular surface epithelia, and other types may be acquired in keratinizing diseases. The intraocular tissues, which have a relatively simple cytoskeleton consisting mainly of vimentin and simple epithelial CK 8 and 18, differ in many details from extraocular ones. The iris and lens epithelium characteristically lack cytokeratins in adults, and the intraocular muscles all have a cytoskeletal profile of their own. The dilator of the iris contains vimentin, desmin and cytokeratins, being an example of triple intermediate filament expression, but the ciliary muscle lacks cytokeratin and the sphincter of the iris is devoid even of vimentin. Conversion from extraocular-type cytoskeletal profile occurs during fetal life. It seems that posttranslational modification of cytokeratins in the eye may also differ from that of extraocular tissues. So far, it has not been possible to reconcile the cytoskeletal profile of intraocular tissues with their specific functional demands, but many theories have been put forward. Systematic search for cytoskeletal elements has also revealed novel cell populations in the human eye. These include transitional cells of the cornea that may represent stem cells on migration, myofibroblasts of the scleral spur and juxtacanalicular tissue that may modulate aqueous outflow, and subepithelial matrix cells of the ciliary body and myofibroblasts of the choroid that may both participate in accommodation. In contrast to the structure and development of the ocular cytoskeleton, changes that take place in ocular disease have not been analysed systematically. Nevertheless, potentially meaningful changes have already been observed in corneal dystrophies (Meesmann's dystrophy, posterior polymorphous dystrophy and iridocorneal endothelial syndrome), degenerations (pterygium) and inflammatory diseases (Pseudomonas keratitis), in opacification of the lens (anterior subcapsular and secondary cataract), in diseases characterized by proliferation of the retinal pigment epithelium (macular degeneration and proliferative vitreoretinopathy), and in intraocular tumours (uveal melanoma). In particular, upregulation of alpha-smooth muscle actin seems to be a relatively general response typical of spreading and migrating corneal stromal and lens epithelial cells, trabecular cells and retinal pigment epithelial cells.

Wnts as kidney tubule inducing factors.

Since the discovery that inductive tissue interactions regulate nephrogenesis, one of the aims has been to identify the molecules that mediate this induction. The small size of embryonic tissue has limited the possibilities to identify the inducers biochemically, even though such efforts were directed to study, e.g. neural induction (for a comprehensive review, Saxén and Toivonen, Primary embryonic induction, Academic Press, London, 1962). The rapid progress in molecular biology made it possible to identify genes from minute amounts of tissue and provided techniques to generate recombinant proteins to assay their action in classic experimental systems. This led to the identification of some signals that are involved in primary and secondary inductive interactions during embryogenesis. Here, we will review evidence suggesting that secreted signaling molecules from the Wnt gene family mediate kidney tubule induction.

Alloimmune injury preceding airway obliteration in porcine heterotopic lung implants: a histologic and immunohistologic study.

BACKGROUND: Obliterative bronchiolitis (OB), the major long-term complication of lung transplantation, has thus far lacked a good large-animal model. Our goal was to develop such a model on the basis of previous rodent models with tracheal implants. METHODS: Fragments of pulmonary tissue with structures of terminal bronchi were subcutaneously transplanted to four random-bred domestic piglets. Each animal received 10 autograft and 10 allograft implants. The histologic findings were graded from 0 to 3 for implants harvested repeatedly over 2 months. RESULTS: In autografts, partial destruction of the respiratory epithelium and gradual luminal obliteration as well as mild damage to the cartilage and the bronchial wall underwent rapid reversal after initial ischemic injury. In the allografts, epithelial destruction and gradual obliteration were total within 14 days, the difference being statistically significant (P<0.05) in both. The histologic features of the obliterative plug were similar to those of human OB. In the allografts, cartilaginous destruction and pericartilaginous inflammation increased gradually to severe levels, significantly worse than in the autografts (P<0.05). Necrosis and inflammation of the bronchial wall were also more severe in the allografts (P<0.05). CONCLUSIONS: At the end of follow-up, all autografts were vital, whereas the allografts were almost totally rejected and were without native structures. All bronchi in the allografts exhibited accelerated obliteration with histologic features characteristic of human OB, thus providing a model for research into OB and its prevention.

Development of cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human eye.

PURPOSE: To analyze the cytoskeletal development in neuroectodermally derived epithelial and muscle cells of the human eye during the second and third trimesters of pregnancy. METHODS: Nine formalin-fixed, paraffin-embedded fetal autopsy eyes from the 13th to 40th week of gestation were studied with eight monoclonal antibodies (mAbs) to intermediate filaments and alpha-smooth muscle actin (alpha SMA) by the avidin-biotinylated-peroxidase complex method. RESULTS: The epithelium of the iris reacted with mAbs Vim 3B4 and V9 to vimentin in all eyes. Reactivity with mAb CAM 5.2 to cytokeratin (CK) 8 and mAb CY-90 and KS-B17.2 to CK 18 disappeared from the iris epithelium by the 28th gestational week. mAb 1A4 to alpha SMA labeled its anterior layer from the 28th week on. mAbs DE-U-10 and D33 to desmin labeled dilator fibers by the 37th week, and focal reactivity for CK 8 and 18 concurrently appeared. The developing iris sphincter reacted for alpha SMA in all eyes. It was labeled increasingly for desmin from the 18th week on, whereas initial reactivity for vimentin gradually disappeared after the 22nd week. mAbs to vimentin, CK 8, and CK 18 labeled the ciliary epithelium and the retinal pigment epithelium in all eyes, except for lack of CK 8 and 18 in the nonpigmented ciliary epithelium at the 13th week. The ciliary muscle reacted uniformly for vimentin and alpha SMA and from the 16th week on for desmin. CONCLUSIONS: The results highlight the individuality of cytoskeletal profiles in the neuroectodermal epithelial and muscle cells of the eye and clarify the formation of the peculiar cytoskeleton typical of the adult human eye. They also offer a framework for detecting cytoskeletal changes in developmental anomalies of the eye after the first trimester of pregnancy.

Autopsy analysis of clinically unilateral exfoliation syndrome.

PURPOSE: To study the pathogenesis of clinically unilateral exfoliation syndrome by localizing exfoliation deposits in involved and fellow eyes during autopsy. METHODS: The formalin-fixed and paraffin-embedded involved and fellow eyes were obtained at autopsy from five patients (age range, 72 to 88 years) with clinically unilateral exfoliation. Exfoliation deposits were identified with monoclonal antibodies (mAb) HNK-1 and NC-1 to the HNK-1 carbohydrate epitope, and with five lectins (Bauhinia purpurea agglutinin, Concanavalin A, Lens culinaris agglutinin, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin I) using the avidin-biotinylated peroxidase complex (ABC) method. RESULTS: Marked exfoliation deposits in all involved eyes, and weak exfoliation deposits in one fellow eye were consistently detected in light microscopic, immunohistochemical, and lectin histochemical examinations. Similarly labeled deposits were present around a population of blood vessels of the iris in every involved and fellow eye. Particularly in fellow eyes, these subendothelial deposits were better visualized with mAbs to the HNK-1 epitope than they were with lectins. In the only fellow eye with early exfoliation, the reactivity around blood vessels was more conspicuous than the exfoliation deposits, whereas the reverse was true in the involved eyes. CONCLUSIONS: Clinically unilateral exfoliation is asymmetric, rather than truly monocular. The findings in fellow eyes suggest that iris blood vessels become abnormal early in the process, even before exfoliation deposits are histopathologically seen in the posterior chamber. Marked asymmetry in exfoliation indicates an influence of modulating local factors that may be internal or external to the eye, and that also may be functional in bilateral exfoliation.

Identification of a novel element in the human eye: the inner connective tissue layer of the ciliary body characterized with antibodies to the HNK-1 epitope.

PURPOSE: To characterize the nature and the developmental distribution of the HNK-1 epitope in the inner connective tissue layer of the human ciliary body, located between the ciliary epithelium and muscle with two monoclonal antibodies to the HNK-1 epitope common to many cell adhesion molecules. METHODS: Nine fetal (gestational age 13-40 wk) and 32 postnatal human eyes (age 3 mo to 78 yr) were studied by immunohistochemistry with monoclonal antibodies HNK-1 and VC1.1 to the HNK-1 epitope. Antibodies to cytoskeletal elements were used to characterize the cells in this region. RESULTS: The HNK-1-immunopositive cells appeared underneath the pigment epithelium of the pars plicata by the 20th gestational week, spread into the pars plana after the 28th week, and reached the ora serrata during the first year of life. The immunoreaction was constantly present in all adult eyes examined; they were sharply demarcated from the iris, ciliary muscle, and choroid. The HNK-1-positive subepithelial layer was not labeled with monoclonal antibodies V9 or Vim 3B4 to vimentin, monoclonal antibodies CAM 5.2 and CY-90 to cytokeratin 8 and 18, or monoclonal antibodies DE-U-10 and D33 to desmin in adult eyes, but was uniformly positive for vimentin in fetal eyes. The HNK-1 epitope was distributed along cell membranes or adjacent extracellular matrix of stromal cells. CONCLUSION: The HNK-1-positive stromal region is a constant and conspicuous element of the human eye that may have a role in structurally stabilizing the ciliary body, perhaps in relation to accommodation or aqueous secretion.

The HNK-1 epitope and the elastic fiber system of the human ciliary body. An immunoelectron microscopic study.

PURPOSE: To localize at the electron microscopic level the cell adhesion-related HNK-1 carbohydrate epitope in the inner connective tissue layer (ICTL) of the human ciliary body. METHODS: Seven specimens representing the pars plicata (age range, 12 to 86 years) and three specimens representing the pars plana (age range, 29 to 71 years) of the ciliary body were sampled at death from eight normal human eyes. Three additional specimens from the pars plicata were taken from three eyes with exfoliation syndrome (age range, 78 to 81 years). All specimens were embedded in LR white resin and studied by postembedding immunogold labeling using two monoclonal antibodies (mAbs) HNK-1 and NC-1 in detecting the HNK-1 epitope. RESULTS: In the ICTL of the pars plicata and the pars plana, mAbs HNK-1 and NC-1 constantly bound to the surface of fibroblast-like cells present in the subepithelial connective tissue matrix. The immunoreaction was localized along the cell membrane, both around the cell body and around its long, slender cytoplasmic processes. Long microfibrillar bundles, which consisted of approximately 10-nm thick microfibrils in close association with these subepithelial matrix cells and elastic fibers, were also labeled. The periphery of elastic fibers was likewise immunolabeled for the HNK-1 epitope. The reaction pattern was essentially the same, regardless of age and presence or absence of exfoliation syndrome. CONCLUSIONS: At the ultrastructural level, the HNK-1 epitope in the ICTL is a common denominator to the subepithelial matrix cells, microfibrillar bundles, and elastic fibers. This suggests that the subepithelial matrix cells secrete this epitope, and that molecules hearing it may be involved in joining these connective tissue elements that structurally stabilize the ciliary body.

Small airway obliteration in a new swine heterotopic lung and bronchial allograft model.

BACKGROUND: We studied a heterotopic large-animal model with obliterative airway lesions caused by allograft rejection. METHODS: Lung fragments (1 cm3) with airways (LB), and 1 to 2 mm diameter bronchi alone (B) were implanted subcutaneously in 11 domestic piglets weighing 20 kg. Six animals each received 40 implants from nonrelated donors without immunosuppression (group A). Another 5 animals had autograft implants (group U). The implants were harvested consecutively for histologic analysis over 3 months in group A and 6 months in group U. RESULTS: In group U, the initial ischemia caused mild to moderate epithelial damage with moderate metaplasia but with a return to normal ciliary epithelium within 1 month. Transient mild luminal obliteration with granulation tissue and mononuclear cells was observed during the first weeks, but after 4 weeks the lumen was patent and filled with mucus. In the bronchial wall, moderate fibrosis developed in LB implants, whereas mild fibrosis was seen in B implants. In group A, the epithelium was totally absent by 2 weeks, and mild inflammation, fibrosis, and destruction of the cartilage with pericartilaginous mononuclear accumulation were observed in the bronchial wall. Small airways were gradually obliterated between days 7 and 21, initially by granulation tissue and mononuclear cells and later by progressive fibrosis. CONCLUSIONS: In this model, autografted airway implants stayed patent for at least 6 months, whereas total luminal obliteration histologically resembling obliterative bronchiolitis developed in allografts within 21 days. Because small airways, including bronchioli, can be transplanted with the use of this model, it may be useful for research into the causes of airway obliteration, which may be relevant to the pathogenesis of obliterative bronchiolitis in lung recipients.

Immunoreactivity of exfoliation material for the cell adhesion-related HNK-1 carbohydrate epitope.

OBJECTIVE: To study the presence of the HNK-1 epitope in exfoliation material. METHODS: Twenty-six formalin-fixed, paraffin-embedded human eyes with exfoliation syndrome and 30 control eyes were studied immunohistochemically with monoclonal antibodies HNK-1 and VC1.1 to the HNK-1 epitope. RESULTS: Exfoliation material reacted consistently with antibodies to the HNK-1 epitope. The zonular lamella of the lens, inner surface of the nonpigmented ciliary epithelium, and inner connective tissue layer of the ciliary body were also labeled, but the lens capsule, epithelium, and zonules were not immunoreactive. Several blood vessels of the iris showed granular immunoreaction beneath the endothelium in all exfoliation eyes and in 11 (37%) of 30 control eyes, representing older age groups. CONCLUSIONS: The zonular lamella, nonpigmented ciliary epithelium, or the inner connective tissue layer may be responsible for the HNK-1 epitope in exfoliation material. Since this epitope is shared by many cell-adhesion molecules, its presence in exfoliation material might be of pathogenetic significance to the formation of the deposits.

Reconstruction with rectus abdominis myocutaneous free flap after orbital exenteration in children.

OBJECTIVE: To present a 1-stage technique for orbital reconstruction after exenteration with the use of myocutaneous rectus abdominis free flap in children. SURGICAL TECHNIQUE: After orbital exenteration, a myocutaneous rectus abdominis free flap with long vascular pedicle is harvested from the abdomen. The flap is transferred to the orbit and the vascular pedicle is passed through an opening made in the lateral orbital wall, where it is anastomosed to superficial temporal vessels. The skin of the flap is trimmed to correspond to the eyelid defect and the incisions are closed. METHODS: After informed consent was obtained, 2 children, 3 and 8 years old, underwent orbital reconstruction with a rectus abdominis free flap after exenteration for orbital rhabdomyosarcoma and orbital osteosarcoma in the setting of retinoblastoma. RESULTS: This technique allowed easy postoperative wound care. Viability of the flap was excellent. The technique provided sufficient volume to fill the orbit, with improved aesthetic results and minimal donor site deformity. CONCLUSIONS: The postoperative care and aesthetic outcome in patients with rectus abdominis free flap after exenteration are much improved over those provided with traditional surgical techniques. This primary reconstruction is recommended for any patient requiring orbital exenteration, but particularly for pediatric patients who tolerate debridement of traditional exenteration sites poorly.

Evaluation of chemoprophylaxis in patients with unilateral retinoblastoma with high-risk features on histopathologic examination.

OBJECTIVES: To identify risk factors for metastatic disease on histopathologic specimens of enucleated eyes from patients with unilateral retinoblastoma, and to evaluate the value of chemoprophylaxis in preventing disease dissemination. METHODS: Medical records from patients with unilateral retinoblastoma who underwent primary enucleation were reviewed at the University of California, San Francisco (1977-1998) and Bascom Palmer Eye Institute, University of Miami, Miami, Fla (1991-1998). All routine histopathologic specimens were reexamined. The extent of tumor invasion into the optic nerve or ocular coats and the prescribed chemoprophylactic regimen were recorded. RESULTS: This retrospective study included 129 patients followed for a median of 54 months. Three patients had tumor invading the sclera. The optic nerve was involved to some extent in 82 patients, 11 of whom had tumor extension beyond the lamina cribrosa. The surgical margin of the optic nerve was involved in an additional 4 patients. The choroid was involved in 43 patients, and was considered massively affected in 12 patients. Anterior segment involvement was observed in 10 patients. Postenucleation chemoprophylaxis was administered to 4 of 4 patients who had tumor cells at the surgical margin of the optic nerve and to 7 of 11 patients with postlaminar disease, all of whom had at least 1 mm of postlaminar tumor extension. External beam radiotherapy was administered to 3/4 and 1/11 of these patients, respectively. Chemoprophylaxis was not administered to patients with choroidal or anterior chamber involvement unless the optic nerve was also involved beyond the lamina cribrosa. One patient with tumor extending to the surgical margin of the optic nerve died of metastatic disease. CONCLUSIONS: Chemoprophylaxis is necessary for patients with tumor extending to the surgical margin of the optic nerve and is likely to be beneficial in preventing metastases in patients with tumor extending beyond the lamina cribrosa. We did not offer chemoprophylaxis to patients with prelaminar optic nerve disease or isolated choroidal involvement, and these patients remained free of disseminated disease.

Characterizing the local oscillatory content of spontaneous cortical activity during mental imagery.

We report on the determination of detailed spectra for simultaneously active sources of spontaneous neuronal activity in humans directly from data recorded with a whole-scalp 122-channel magnetometer array. Subjects rested with eyes open and performed two contrasting mental imagery tasks: the imagination of the self-performance of a motor activity and the silent generation of a chain of words. A novel analysis technique, frequency-domain signal-space projection (FDSSP) was utilized to determine the temporal and spectral characteristics of spontaneous brain activity at specific cortical sites. Although intersubject differences were significant, spectra for individual subjects contained task-dependent features which were reproducible over successive 20-s epochs. This result supports the concept of multiple sources of spontaneous cortical activity and suggests that detailed spectra of localized oscillatory activity obtained non-invasively with magnetoencephalographic arrays may provide a useful characterization of cortical involvement in mental imagery.

Visual motion activates V5 in dyslexics.

A recent functional magnetic resonance imaging (fMRI) study concluded that the motion-specific visual area V5 is not activated in dyslexic subjects. We report here opposing evidence based on whole-scalp neuromagnetic recordings. Apparent-motion stimuli elicited similar activation of V5 in both dyslexic and control subjects, with a trend for longer latencies in dyslexics. Both high- and low-contrast stimuli activated the V5 region in dyslexics. The lack of significant blood flow changes despite modified neuronal synchrony would explain the absence of fMRI signals and the presence of neuromagnetic signals in dyslexic subjects.

Activation trace lifetime of human cortical responses evoked by apparent visual motion.

Visually evoked magnetoencephalographic responses were recorded from 11 healthy humans to 1.1 x 1.1 degrees oblique gratings moving quickly 0.2 degree rightwards and back once every 0.2-6.4 s. The aim was to study the duration of sensory memory in the motion-specific visual cortex called V5. Responses from the V5 region peaked at 140-180 ms after stimulus onset. Signal-to-noise ratio allowed source identification in eight subjects: bilaterally in four and unilaterally in four. The response strength as a function of interstimulus interval determined an activation trace lifetime, reflecting how long the preceding stimuli affect the response to the following stimulus, i.e. how long the V5 cortex "remembers' each stimulus. The lifetimes varied interindividually from 0.4 to 1.4 s, but were within 0.1 s in the hemispheres of the four subjects with bilaterally identified sources.

Dynamical organisation of the human visual system revealed by lifetimes of activation traces.

Magnetic source imaging reveals a dynamical organisation of visual cortical areas suggesting that the participation of local memories is an essential component of visual information processing. Response recovery studies provide evidence that each responding cortical area supports a memory function with a well-defined lifetime. The areas fell into two groups, the earliest in occipital lobes with lifetimes ranging from 0.1 to 0.6 s, and the later ones in temporal, parietal, and frontal areas with lifetimes ranging from 7 to 30 s. Also, within each group the areas responding later tended to have longer lifetimes.

Lectin and immunohistochemical comparison of glycoconjugates in the conjunctiva of patients with and without exfoliation syndrome.

AIMS--The study was carried out to search for labelling similar to that of intraocular exfoliation material in the conjunctiva by light microscopy using lectin and immunohistochemistry. METHODS--Ten formalin fixed and paraffin embedded conjunctival biopsy specimens both from patients with and without exfoliation syndrome were studied with a panel of 11 lectins and with three monoclonal antibodies to the HNK-1 carbohydrate epitope, all of which react with intraocular exfoliation material. RESULTS--The lectin binding profile was essentially the same in specimens from patients with and without exfoliation syndrome. The superficial epithelium reacted similarly with Phaseolus vulgaris (PHA-E), Caragana arborescens (CAA), Helix pomatia (HPA), concanavalin A (ConA), and wheat germ (WGA) agglutinins. Binding was also detected with peanut (PNA) and Bauhinia purpurea (BPA) agglutinins, particularly in patients with exfoliation. The basement membrane generally reacted with Ricinus communis (RCA-I), PHA-E, Vicia villosa (VVA), ConA, and Lens culinaris (LCA) agglutinins. The stroma was weakly labelled with RCA-I, PHA-E, ConA, and LCA. Lectin binding to the vascular endothelium was moderate with RCA-I, PHA-E, CAA, ConA, LCA, and WGA. Inconsistent labelling was also detected with PNA, BPA, and Erythrina cristagalli agglutinin (ECA). The subendothelial region reacted weakly but consistently with PHA-E, ConA, and LCA, and inconsistently with PNA. Pretreatment with neuraminidase did not change that pattern. Antibodies to the HNK-1 epitope reacted only with myelinated stromal nerve branches. CONCLUSION--No evidence of abnormal deposits in any specimen was found. The carbohydrate composition of intraocular exfoliation material may differ from that of exfoliation-like fibres often detected in the conjunctiva by electron microscopy.

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina.

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.

Resin modified glass ionomer cements. In vitro microleakage in direct class V and class II sandwich restorations.

Microleakage of conventional chemically cured and resin modified glass ionomer cements (GIC), a compomer and a composite resin was studied in vitro in direct class V and class II GIC/composite sandwich fillings. None of the restorative techniques investigated completely resisted microleakage at both the occlusal and gingival margins. The dentinal gingival margins exhibited the highest leakage pattern. The compomer and the resin modified GIC showed a better seal than the chemically cured GIC's. The use of a liner as separating agent between composite resin and GIC in the class II sandwich fillings did not improve the seal. The results suggest that the initial bond obtained immediately after light curing of the resin modified GIC's is stronger than that for chemically cured GIC's. The contradictory results of dye leakage studies reported in the literature are discussed and in vivo evaluations are suggested necessary to predict clinical performance.